Tobacco-smoking is the single major cause of cancer mortality in the US, and is a risk factor for a number of cancers including lung, upper aero-digestive tract, bladder and pancreas. One of the most powerful carcinogens in tobacco smoke is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK is bioactivated to potent electrophiles that react to form methyl and 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts. Role of methyl-DNA adducts in carcinogenesis have been well characterized and the current paradigm is that methyl- DNA adducts are more important than POB-DNA adducts in the etiology of tobacco-induced cancers. However, recently it was found that O2-POB-dT adduct is the most persistent POB adduct in NNK-treated rodents. Our preliminary results show that O2-POB-dT is inefficiently repaired in human cells and is mutagenic in SOS-induced E. coli and mammalian cells. The objective of this application is to determine the mechanisms by which O2-POB-dT forms mutations and is repaired in mammalian cells. These goals will be examined in two specific aims: (1) to determine the polymerases involved in accurate and mutagenic bypass of O2-POB-dT and (2) to determine the mechanisms by which the POB-adducts are repaired. The polymerases involved in the bypass will be determined in cells in which specific polymerases are down regulated by siRNA, and in vitro with purified polymerases. The role of strand switching during translesion synthesis will be examined with cell- free extracts. The repair mechanisms will be evaluated by a combination of ex vivo and in vitro experiments. The roles of NER and BER will be evaluated ex vivo using cells with deficient repair proteins using a HPLC- MS/MS assay to measure levels of DNA adducts. The repair of O2-POB-dT via NER and BER will examined in vitro using synthetic oligodeoxynucleotides. The role of transcription-coupled NER for O6-POB-dG and O2- POB-dT will be probed with a novel modified host cell reactivation. PUBLIC HEALTH RELEVANCE: One way by which tobacco smoke causes cancer is by damaging the DNA in the cell. If the damage is not repaired then when the cell replicates its DNA mistakes (mutations) are made that can lead to cancer. The goal of this project is to understand how the cell repairs and replicates an important class of DNA damage.